Antimicrobial fusion peptides

ABSTRACT

Antimicrobial peptides, compositions and methods are described that are useful for treating infectious disease, including those caused by drug resistant Gram-negative bacteria (e.g., Pseudomonas and Acinetobacter) and parasite-caused disease such as malaria. The peptides include a modular kinocidin gamma-core connected directly, or through a short spacer, to a kinocidin C-terminal alpha-helix.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 15/579,926, filed Jul. 2,2018, now allowed, which is a U.S. National Stage Application under 35U.S.C. § 371 of International Application No. PCT/US2016/035896, filedJun. 3, 2016, which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 62/171,868, filed on Jun. 5, 2015, eachof which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2018, isnamed 213137-US_ST25.txt and is 8,192 bytes in size.

BACKGROUND

Infection is an increasingly serious problem to public health, fromintensive care units (ICUs), to trauma and wound infection, toinfections among the immune compromised. Infection is associated withunacceptable morbidity, mortality, and cost. According to the results ofthe recent EPIC II studies, infected patients have much higher hospitalmortality rates, up to 33.1% versus 14.8% for non-infected patients.Despite the recent media attention focused on methicillin-resistantStaphylococcus aureus (MRSA), the looming threat in MDR pathogens overthe last decade has increasingly been the rise of Gram-negativepathogens Pseudomonas aeurginosa (PA) or Acinetobacter baumannii (AB).

Such pathogens are isolated in 62% of patients in ICU settings, versus47% for Gram-positive pathogens. Pseudomonas aeruginosa is one of themost common life-threatening pathogens in public health, particularlyamong immune compromised or immune suppressed patients. Infectionscaused by multi-drug-resistant Pseudomonas aeurginosa (MDRPA) can occurin any person, including hospital acquired pneumonia,catheter-associated infections and bacteremia, sepsis, respiratoryinfections, penetrating, crushing, or wound infections, andcommunity-acquired infections of the eyes, ears, or sinuses fromwaterborne sources. Recent, population-based data of nosocomialinfections caused by MDRPA in Europe identified an incidence of 126cases per 100,000 individuals.

Likewise, multi-drug-resistant Acinetobacter baumannii (MDRAB) is anincreasingly urgent and unaddressed pathogen responsible forlife-threatening infections, particularly in ICU settings, includingventilator associated pneumonia, bacteremia, surgical site infection,and sepsis. Treating such infections caused by MDRAB is difficult due toresistance to multiple antibiotics, including carbapenems, andattributable mortality rates are high.

Infections caused by MDRPA and MDRAB are virtually always resistant tomany classes of antibiotics, and commonly resistant all antibiotics inthe medical formulary. Thus, there are increasing reports of trulypan-resistant infections due to these organisms which cannot be treatedwith any available antibiotic or combination.

Correlating with a highly antibiotic-resistant nature, infections causedby MDRPA or MDRAB are associated with high mortality rates. For example,MDRPA is associated with increased risk of mortality from sepsis, and isa significant predictor of ICU mortality. Finally, there is a concerningabsence of new antibiotics or those in development that have novelmechanisms of action against these agents. For example, even a recentlyapproved antibiotic, tigecycline, is inactive vs. MDRPA. Of greaterconcern, few if any antibiotics currently in phase II or later clinicaltrials has improved efficacy against MDRPA or MDRAB as compared withexisting agents. These alarming trends underscore the desperate need fornew strategies to treat antibiotic-resistant and deadly Gram-negativeinfections.

Another wide-spread infectious disease is malaria. Uncomplicated malariamay be treated with oral medications. The current treatment for P.falciparum infection is the use of artemisinins in combination withother antimalarials (known as artemisinin-combination therapy, or ACT),which may decrease resistance to any single drug component. Theseadditional antimalarials include: amodiaquine, lumefantrine, mefloquineor sulfadoxine/pyrimethamine. Another recommended combination isdihydroartemisinin and piperaquine. ACT is about 90% effective when usedto treat uncomplicated malaria. Infection with P. vivax, P. ovale or P.malariae is usually treated without the need for hospitalization.Treatment of P. vivax requires both treatment of blood stages (withchloroquine or ACT) and clearance of liver forms with primaquine.Recommended treatment for severe malaria is the intravenous use ofantimalarial drugs. For severe malaria, artesunate is superior toquinine in both children and adults.

Despite potential benefits of combination therapy, drug resistance tovarious malaria treatments poses a growing problem in the 21st-century.Resistance is now common against all classes of antimalarial drugs apartfrom artemisinins. Treatment of resistant strains became increasinglydependent on this class of drugs. The cost of artemisinins limits theiruse in the developing world. Malaria strains found on theCambodia-Thailand border are resistant to combination therapies thatinclude artemisinins, and may therefore be untreatable. Exposure of theparasite population to artemisinin monotherapies in subtherapeutic dosesfor over 30 years and the availability of substandard artemisininslikely drove the selection of the resistant phenotype.

SUMMARY

The present disclosure describes antimicrobial peptides, compositionsand methods that are useful for treating infectious diseases, inparticular those caused by drug resistant Gram-negative bacteria (e.g.,Pseudomonas and Acinetobacter) and parasite-caused diseases such asmalaria. Such antimicrobial peptides, compositions and methods are alsosuitable for treating fungus or virus-caused infections, withoutlimitation. In some embodiments, the peptides include a modularkinocidin gamma-core connected directly, or through a short spacer, to akinocidin C-terminal alpha-helix.

In one embodiment, the present disclosure provides an isolated peptidecomprising (1) a first fragment consisting of the amino acid sequence ofSEQ ID NO:2 (CPTAQLIATLKNGRKICLDLQ) or a first amino acid sequencehaving at least 85% sequence identity to SEQ ID NO: 2; and (2) a secondfragment consisting of the amino acid sequence of SEQ ID NO:3(ALYKKFKKKLLKSLKRLG) or a second amino acid sequence having at least 85%sequence identity to SEQ ID NO: 3, wherein the first fragment and thesecond fragment are connected directly or connected through a spacerthat is 10 amino acids or fewer in length.

In some aspects, the first fragment is at the N-terminal end of thesecond fragment. In some aspects, the first fragment and the secondfragment are connected directly. In some aspects, the first fragment andthe second fragment are connected through the spacer. In some aspects,the spacer is 5 amino acids or fewer in length. In some aspects, thespacer comprises one or more glycine.

In some aspects, the first fragment consists of the amino acid sequenceof SEQ ID NO:4 (CPTAQLIATLKNGRKICLDLQP). In some aspects, the firstfragment consists of the amino acid sequence of SEQ ID NO:5(CPTAQLIATLKNGRKICLDLQAP). In some aspects, the first fragment consistsof the amino acid sequence of SEQ ID NO:6 (CPTAQLIATLKNGPKICLDLQ).

In some aspects, the peptide comprises the amino acid sequence of SEQ IDNO:1 (CPTAQLIATLKNGRKICLDLQALYKKFKKKLLKSLKRLG). In some aspects, thepeptide comprises the amino acid sequence of SEQ ID NO:13

(CPTAQLIATLKNGRKICLDLQAALYKKFKKKLLKSLKRLG). In some aspects, the peptidecomprises the amino acid sequence of SEQ ID NO:14(CPTAQLIATLKNGRKICLDLQPALYKKFKKKLLKSLKRLG). In some aspects, the peptidecomprises the amino acid sequence of SEQ ID NO:15(CPTAQLIATLKNGRKIPLDLQALYKKFKKKLLKSLKRLG).

In some aspects, the peptide is not longer than 100 amino acids inlength. In some aspects, the peptide is not longer than 75 amino acidsin length. In some aspects, the peptide is not longer than 50 aminoacids in length. In some aspects, the peptide has antimicrobialactivity.

In one embodiment, provided is a polynucleotide comprising a nuclei acidsequence encoding the peptide of any one of the peptides of the presentdisclosure. In another embodiment, provided is a composition comprisinga peptide of the present disclosure and a pharmaceutically acceptablecarrier. In some aspects, the composition further comprises anantimicrobial agent. In some aspects, the antimicrobial agent isselected from the group consisting of imipenem, ceftazidime, colistin,chloroquine, artemisinin, vancomycin and daptomycin.

Yet another embodiment provides a method of treating an infection in apatient in need thereof, comprising administering to the patient aneffective amount of the peptide or composition of the presentdisclosure. In some aspects, the infection is caused by a Gram-negativebacterium. In some aspects, the infection is multi-drug-resistant. Insome aspects, the infection is caused by multidrug-resistant Pseudomonasaeruginosa (MDRPA) or multidrug-resistant Acinetobacter baumannii(MDRAB).

In some aspects, the patient suffers from a disease or conditionselected from the group consisting of wound abscess, catheter biofilm,pneumonia, and bacteremia. In some aspects, the infection is caused by aparasite. In some aspects, the patient suffers from malaria. In someaspects, the administration is intravenous or topical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B illustrate RP-1 and γ-RP-1 composite mechanisms of actionagainst MDRPA and MDRAB in vitro.

FIG. 2 shows the efficacy of RP-1 and γ-RP-1 against reference strainsof MDRAB by radial diffusion in vitro.

FIG. 3 presents comparative MICs against reference strains of MDRABusing CLSI assay methods.

FIG. 4 shows peptide efficacies against a reference strain of MDRAB inhuman blood matrices.

FIG. 5 presents charts showing mechanisms of peptide or antibioticaction (MOA) against a reference strain of MDRAB in vitro.

FIG. 6 compares peptide or imipenem efficacy against a reference strainof MDRAB in neutropenic pneumonia in vivo.

FIG. 7 compares the efficacy of peptide or imipenem against a referencestrain of MDRAB in representative tissues (lung, spleen) in vivo.

FIG. 8-11 present charts showing the efficacy of different peptidesagainst a panel of Gram-negative human pathogens under differingconditions of pH (5.5 or 7.5) in vitro.

FIG. 12 shows a curve demonstrating the quantitative in vitroantimalarial activity of γ-RP-1 on 3D7 P. falciparum.

FIG. 13 shows in vitro tolerability data of γ-RP-1 on Human BrainEndothelial Cells (HBECs).

FIG. 14 shows the in vivo antimalarial efficacy of γ-RP-1 versuschloroquine on P. berghei ANKA luciferase infected Swiss Webster mice.

FIG. 15 shows the in vivo antimalarial activity of γ-RP-1 onLuciferase-expressing P. berghei ANKA (single dose).

FIG. 16A-B present charts showing the efficacy of different peptidesagainst a panel of Gram-negative human pathogens under differingconditions of pH (5.5 or 7.5) in vitro.

FIG. 17A-C present charts showing the efficacy of different peptidesagainst a panel of Gram-negative human pathogens under differingconditions of pH (5.5 or 7.5) in vitro.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of peptides.

1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. As used herein the followingterms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) claimed. “Consisting of” shallmean excluding more than trace elements of other ingredients andsubstantial method steps. Embodiments defined by each of thesetransition terms are within the scope of this disclosure.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including range, indicatesapproximations which may vary by (+) or (−) 10%, 5% or 1%.

As used herein, the term “sequence identity” refers to a level of aminoacid residue or nucleotide identity between two peptides or between twonucleic acid molecules. When a position in the compared sequence isoccupied by the same base or amino acid, then the molecules areidentical at that position. A peptide (or a polypeptide or peptideregion) has a certain percentage (for example, at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 83%, or at least about 85%, or atleast about 90%, or at least about 95%, or at least about 98% or atleast about 99%) of “sequence identity” to another sequence means that,when aligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. It is noted that, for any sequence(“reference sequence”) disclosed in this application, sequences havingat least about 60%, or at least about 65%, or at least about 70%, or atleast about 75%, or at least about 80%, or at least about 83%, or atleast about 85%, or at least about 90%, or at least about 95%, or atleast about 98% or at least about 99% sequence identity to the referencesequence are also within the disclosure.

Likewise, the present disclosure also includes sequences that have one,two, three, four, or five substitution, deletion or addition of aminoacid residues or nucleotides as compared to the reference sequences.

In any of the embodiments described herein, analogs of a peptidecomprising any amino acid sequence described herein are also provided,which have at least about 80%, or at least about 83%, or at least about85%, or at least about 90%, or at least about 95%, or at least about98%, or at least about 99% sequence identity to any of reference aminoacid sequences. In some embodiments, the analogs include one, two,three, four, or five substitution, deletion or addition of amino acidresidues as compared to the reference sequences. In some embodiments,the substitution is a conservative substitution.

As is well-known in the art, a “conservative substitution” of an aminoacid or a “conservative substitution variant” of a peptide refers to anamino acid substitution which maintains: 1) the secondary structure ofthe peptide; 2) the charge or hydrophobicity of the amino acid; and 3)the bulkiness of the side chain or any one or more of thesecharacteristics. Illustratively, the well-known terminologies“hydrophilic residues” relate to serine or threonine. “Hydrophobicresidues” refer to leucine, isoleucine, phenylalanine, valine oralanine, or the like. “Positively charged residues” relate to lysine,arginine, ornithine, or histidine. “Negatively charged residues” referto aspartic acid or glutamic acid. Residues having “bulky side chains”refer to phenylalanine, tryptophan or tyrosine, or the like. A list ofillustrative conservative amino acid substitutions is given in Table A.

TABLE A For Amino Acid Replace With Alanine D-Ala, Gly, Aib, β-Ala,L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn,Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr,L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-AspGlutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala,D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu,Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, He LysineD-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp,D-Trp Proline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-CysThreonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val TyrosineD-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

Alternatively, non-limiting examples of conservative amino acidsubstitutions are provided in the table below, where a similarity scoreof 0 or higher indicates conservative substitution between the two aminoacids.

C G P S A T D E N Q H K R V M I L F Y W W −8 −7 −6 −2 −6 −5 −7 −7 −4 −5−3 −3 2 −6 −4 −5 −2 0 0 17 Y 0 −5 −5 −3 −3 −3 −4 −4 −2 −4 0 −4 −5 −2 −2−1 −1 7 10 F −4 −5 −5 −3 −4 −3 −6 −5 −4 −5 −2 −5 −4 −1 0 1 2 9 L −6 −4−3 −3 −2 −2 −4 −3 −3 −2 −2 −3 −3 2 4 2 6 I −2 −3 −2 −1 −1 0 −2 −2 −2 −2−2 −2 −2 4 2 5 M −5 −3 −2 −2 −1 −1 −3 −2 0 −1 −2 0 0 2 6 V −2 −1 −1 −1 00 −2 −2 −2 −2 −2 −2 −2 4 R −4 −3 0 0 −2 −1 −1 −1 0 1 2 3 6 K −5 −2 −1 0−1 0 0 0 1 1 0 5 H −3 −2 0 −1 −1 −1 1 1 2 3 6 Q −5 −1 0 −1 0 −1 2 2 1 4N −4 0 −1 1 0 0 2 1 2 E −5 0 −1 0 0 0 3 4 D −5 1 −1 0 0 0 4 T −2 0 0 1 13 A −2 1 1 1 2 S 0 1 1 1 P −3 −1 6 G −3 5 C 12

As used herein, the term “composition” refers to a preparation suitablefor administration to an intended patient for therapeutic purposes thatcontains at least one pharmaceutically active ingredient, including anysolid form thereof. The composition may include at least onepharmaceutically acceptable component to provide an improved formulationof the compound, such as a suitable carrier. In certain embodiments, thecomposition is formulated as a film, gel, patch, or liquid solution.

As used herein, the term “pharmaceutically acceptable” indicates thatthe indicated material does not have properties that would cause areasonably prudent medical practitioner to avoid administration of thematerial to a patient, taking into consideration the disease orconditions to be treated and the respective route of administration. Forexample, it is commonly required that such a material be essentiallysterile.

2. Antimicrobial Peptides

Meritorious efforts to translate antimicrobial activity of classicalantimicrobial peptides (e.g., magainins or defensins) into novelsystemic anti-infectives have met with considerable difficulty over thepast decade. The predominant barriers yet to be overcome are toxicityand durable efficacy in the bloodstream. The activity of theseantimicrobial peptides is typically restricted to phagolysosomes or torelatively inert epithelial surfaces, such as skin or mucosa. Beyondthese contexts, such peptides are typically toxic, inactive, ordegraded.

Kinocidins are naturally liberated into the bloodstream to exert theirantimicrobial effects in the context of blood, plasma and serum. TheC-terminal α-helix of kinocidins, when isolated, possess anti-infectiveactivities that are similar to or even stronger than the naturalkinocidins. RP-1 (ALYKKFKKKLLKSLKRLG; SEQ ID NO:3) is a consensussequence obtained from the α-helix motifs of multiple kinocidinproteins. As shown in the experiments, RP-1 has antimicrobial activityagainst a wide range of human pathogens.

Another common structural element of the kinocidins is amultidimensional γ-core signature (see, e.g., Yount and Yeaman, ProcNatl Acad Sci USA. 101:7363-8, 2004). There was no report concerning theantimicrobial activity of the γ-core. Surprisingly, however, the presentexperimental examples demonstrate that γ-RP-1 (SEQ ID NO: 1), whichfuses the RP-1 peptide to a γ-core of the CXCL4 protein, exhibitedpotent antimicrobial activities against a wide range of human pathogens,including Gram-negative bacteria and parasites. Even more surprisingly,γ-RP-1 is more potent than RP-1 and other comparative antimicrobialpeptides, and is efficacious among a wider range of microorganisms, canexhibit activities in a wider variety of environments, and appears tohave therapeutic tolerability. Importantly, the γ-RP-1 molecule exhibitsgreater efficacy against microbial pathogens in human blood than RP-1.

In accordance with one embodiment of the present disclosure, provided isa fusion peptide that includes RP-1 (SEQ ID NO: 3) or a variant thereofand the γ-core of CXCL4 (CPTAQLIATLKNGRKICLDLQ, SEQ ID NO: 2) or avariant thereof.

A variant of RP-1, as used herein, refers to a peptide that has acertain level of sequence identity to RP-1, or a peptide that can bemade by modifying the RP-1 peptide with one, two or three amino acidaddition, deletion and/or substitution (e.g., conservative substitutionsas illustrated in Table A). The level of sequence identity, in oneaspect, is at least about 80%. In another aspect, the level of sequenceidentify is at least about 85%, at least about 90%, or at least about95%.

A variant of CXCL4 γ-core (SEQ ID NO: 2), as used herein, refers to apeptide that has a certain level of sequence identity to the γ-core, ora peptide that can be made by modifying the γ-core with one, two orthree amino acid addition, deletion and/or substitution (e.g.,conservative substitutions as illustrated in Table A). The level ofsequence identity, in one aspect, is at least about 80%. In anotheraspect, the level of sequence identify is at least about 85%, at leastabout 90%, or at least about 95%.

In one aspect, addition of one, two or three amino acids can be at theN- or C-terminal end of the peptide. For instance, variants of theγ-core can include CPTAQLIATLKNGRKICLDLQP (SEQ ID NO: 4),CPTAQLIATLKNGRKICLDLQAP (SEQ ID NO: 5) and CPTAQLIATLKNGRKICLDLQA (SEQID NO: 7).

Addition, deletion or substitution does not need to be at the end of thepeptide, however. For instance, even though Cys1 and Cys17 of SEQ ID NO:2 form a disulfide bridge, substitution of Cys17 with a proline (i.e.,CPTAQLIATLKNGRKIPLDLQ; SEQ ID NO: 8) did not impact the activity of thefusion peptide. Likewise, CPTAQLIATLKNGPKICLDLQ (SEQ ID NO: 6) is also asuitable variant of the γ-core.

A linker can optionally be included between the γ-core and RP-1, whichis preferably 10 amino acids or fewer in length. In some aspects, thespacer is 9, 8, 6, 5, 4, 3, 2 amino acids in length or shorter. Thespacer can include any amino acids, such as Ala, Pro, Cys, and Gly.

It is contemplated that the γ-core and RP-1 within the fusion peptidecan be at either orientation, i.e., the RP-1 fragment can be at the N-or C-terminal end of the γ-core. The total length of the fusion peptideis preferably not longer than 100 amino acids in length, and morepreferably not longer than 90, 85, 80, 75, 70, 65, 60, 55, or 50 aminoacids in length.

In some embodiments, the fusion peptide has antimicrobial activity.

In some embodiments, the peptides of the present disclosure include SEQID NO:1 and peptides that are modified from SEQ ID NO:1 by includingone, two or three substitution, insertion/addition, deletion, or thecombination therefore. In one aspect, the insertion is with amino acidresidues with small side chains such as glycine. In one aspect, at leastone insertion is between amino acids 20 and 21, between amino acids 21and 22, or between amino acids 22 and 23. In one aspect, such insertionat a particular location is one, two or three amino acids.

In some embodiments, the peptides of the present disclosure are at least35 amino acid in length, or alternatively at least 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 65, 70, 75, or 80 amino acids. In some embodiments, the peptidesof the present disclosure are not longer than 80 amino acids, or notlonger than 75, 70, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49,48, 47, 46, 45, 44, 43, 42, 41, or 40 amino acids.

In some embodiments, the peptides may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

The peptides may be conjugated or fused to a therapeutic agent, whichmay include detectable labels such as radioactive labels, animmunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivetherapeutic or diagnostic agent, a cytotoxic agent, which may be a drugor a toxin, an ultrasound enhancing agent, a non-radioactive label, acombination thereof and other such agents known in the art. The peptidescan be detectably labeled by coupling it to a chemiluminescent compound.The presence of the chemiluminescent-tagged antigen-binding polypeptideis then determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of particularlyuseful chemiluminescent labeling compounds are luminol, isoluminol,theromatic acridinium ester, imidazole, acridinium salt and oxalateester.

3. Synthesis of Antimicrobial Peptides

The peptides described herein can be ordered from a commercial source orpartially or fully synthesized using methods well known in the art(e.g., chemical and/or biotechnological methods). In certainembodiments, the peptides are synthesized according to solid phasepeptide synthesis protocols that are well known in the art. In anotherembodiment, the peptide is synthesized on a solid support according tothe well-known Fmoc protocol, cleaved from the support withtrifluoroacetic acid and purified by chromatography according to methodsknown to persons skilled in the art. In other embodiments, the peptideis synthesized utilizing the methods of biotechnology that are wellknown to persons skilled in the art. In one embodiment, a DNA sequencethat encodes the amino acid sequence information for the desired peptideis ligated by recombinant DNA techniques known to persons skilled in theart into an expression plasmid (for example, a plasmid that incorporatesan affinity tag for affinity purification of the peptide), the plasmidis transfected into a host organism for expression, and the peptide isthen isolated from the host organism or the growth medium, e.g., byaffinity purification.

The peptides can be also prepared by using recombinant expressionsystems. Generally, this involves inserting the nucleic acid moleculeinto an expression system to which the molecule is heterologous (i.e.,not normally present). One or more desired nucleic acid moleculesencoding a peptide of the disclosure may be inserted into the vector.When multiple nucleic acid molecules are inserted, the multiple nucleicacid molecules may encode the same or different peptides. Theheterologous nucleic acid molecule is inserted into the expressionsystem or vector in proper sense (5′→3′) orientation relative to thepromoter and any other 5′ regulatory molecules, and correct readingframe.

Purified peptides may be obtained by several methods. The peptide ispreferably produced in purified form (preferably at least about 80% or85% pure, more preferably at least about 90% or 95% pure) byconventional techniques. Depending on whether the recombinant host cellis made to secrete the peptide into growth medium (see U.S. Pat. No.6,596,509 to Bauer et al., which is hereby incorporated by reference inits entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

4. Antimicrobial Compositions and Formulations

Compositions and formulations that include any one or more of thepeptides as disclosed herein are also provided. In one embodiment, thecomposition includes any one or more of the peptides and apharmaceutically acceptable carrier.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions of thedisclosure. Pharmaceutically acceptable carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

The pharmaceutical compositions of the disclosure can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders,injections, emulsions, elixirs, suspensions or solutions. Formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Pharmaceutical formulations may be prepared as liquid suspensions orsolutions using a sterile liquid, such as oil, water, alcohol, andcombinations thereof. Pharmaceutically suitable surfactants, suspendingagents or emulsifying agents, may be added for oral or parenteraladministration. Suspensions may include oils, such as peanut oil, sesameoil, cottonseed oil, corn oil and olive oil. Suspension preparation mayalso contain esters of fatty acids, such as ethyl oleate, isopropylmyristate, fatty acid glycerides and acetylated fatty acid glycerides.Suspension formulations may include alcohols, such as ethanol, isopropylalcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, suchas poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil andpetrolatum, and water may also be used in suspension formulations.

The compositions of this disclosure are formulated for pharmaceuticaladministration to a mammal, preferably a human being. Suchpharmaceutical compositions of the disclosure may be administered in avariety of ways, preferably parenterally.

Sterile injectable forms of the compositions of this disclosure may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

In addition to dosage forms described above, pharmaceutically acceptableexcipients and carriers and dosage forms are generally known to thoseskilled in the art and are included in the disclosure. It should beunderstood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific peptide employed, the age, body weight, generalhealth, sex and diet, renal and hepatic function of the patient, and thetime of administration, rate of excretion, drug combination, judgment ofthe treating physician or veterinarian and severity of the particulardisease being treated.

In some embodiments, the composition can further include a secondaryantimicrobial agent. Non-limiting examples of such agents includeimipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycinand daptomycin.

5. Methods

Methods of using the peptides, compositions and formulations of thepresent disclosure are also described. In one embodiment, the methodsare for preventing or treating an infection of a microorganism. Themicroorganism can be a bacterium, such as a Gram-negative bacterium, ora parasite (e.g., malaria-causing microorganisms). In some embodiments,the infection is multi-drug-resistant, such as one caused bymultidrug-resistant Pseudomonas aeruginosa (MDRPA) ormultidrug-resistant Acinetobacter baumannii (MDRAB).

The peptides, compositions and formulations are also useful for treatinga disease or condition associated with an infection, such as woundabscess, catheter biofilm, pneumonia, and bacteremia.

In some embodiments, the treatment methods further includeadministration, concurrently or sequentially, of a second secondaryantimicrobial agent. Non-limiting examples of such agents includeimipenem, ceftazidime, colistin, chloroquine, artemisinin, vancomycinand daptomycin.

The peptides, compositions and formulations of the disclosure may beadministered to the systemic circulation via parental administration.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. However, in cases where the infection is local(e.g., on the skin), the composition may be administered locally, suchas topically.

EXAMPLES Example 1. In Vitro Mechanistic Study

This example tested the antibacterial activity of two peptides, RP-1 andγ-RP-1, in vitro and assessed the multi-parametric mechanisms of action(MOA) of these peptides.

RP-1 has a nominal sequence of N-ALYKKFKKKLLKSLKRLG-C(SEQ ID NO:3) andis a consensus sequence obtained from the α-helix motifs of multiplekinocidin proteins. γ-RP-1 (SEQ ID NO: 1) is a fusion that furtherincludes, at the N-terminus of RP-1, a portion of the γ-core of theCXCL4 protein. The γ-core has not been shown to have anti-infectiveactivities.

RP-1 and γ-RP-1 mechanisms were evaluated using a multi-parametric flowcytometry platform optimized to MDRPA and MDRAB. Included in theseinvestigations were assessments of microbicidal vs. microbiostatickinetics, membrane injury, energy decoupling, and mechanisms of ancientprogrammed cell death pathways (caspase/metacaspase induction andphosphatidylserine-like lipid accessibility). Mechanisms of action weredetermined alone and in combination with the conventional antibiotics(e.g., imipenem, ceftazidime, or colistin).

FIGS. 1A and 1B illustrate the mechanisms of action (MOA) of RP-1 andγ-RP-1, assessed alone and combined with conventional antibioticsagainst prototypic strains of MDRPA (47085 or PA-01) and MDRAB (19606),in vitro by flow cytometry. Parameters evaluated: cell size (forwardscatter [FSC]; osmostasis); cytoplasmic refractivity (side scatter[SSC]; DNA/RNA condensation); cellular/membrane energetics (Δψ; [ENR]);cell membrane permeability (PRM); phosphatidylserine accessibility([PSA]; programmed cell death pathway); caspase or metacaspase induction([CSP]; programmed cell death pathway).

Increases in parameters measured are indicated by a red (gray inencircled areas) spectral shift, while decreases in parameters shifttoward a violet spectrum (black in the top middle to left area); green(background gray) indicates no significant change from baseline. Thesedata emphasize distinct, specific mechanistic signatures ofmetapeptides. Each graph integrates more than 1.2 million data points,generated using a multi-color flow cytometry and analyticalmethodologies.

This example compares the metapeptides (RP-1 and γ-RP-1) alone, relevantstandard antibiotics alone (imipenem, ceftazidime, colistin), versuscombinations of metapeptides plus antibiotics. The study assessedmetapeptide impact on multiple aspects of target cell function:osmostasis, cytoplasmic/nucleic acid condensation, energetics, membranepermeabilization, phosphatidyl serine-like lipid turnover andmetacaspase or caspase-like activation (surrogate biomarkers ofprogrammed cell death). The results show that 1) RP-1 and γ-RP-1 havespecific mechanisms and are not indiscriminant membrane detergents; 2)mechanisms of action differ considerably from those of conventionalantibiotics; and 3) γ-RP-1 and RP-1 mechanisms differ against MDRPA vs.MDRAB.

Example 2. Efficacy of Intravenous Kinocidinsin a Neutropenic MurineModel of Multi-Drug-Resistant Acinetobacter baumannii Pneumonia

This example tested the efficacy of RP-1 and γ-RP-1 in a neutropenicmurine model of multi-drug-resistant Acinetobacter baumannii pneumonia.

Methods and Materials

Bacterial strains and sources used in this example are listed below:

Study Strains Human Source AB19606 Blood/Urine AB17978 MeningitisAB-HUMC-1 Bacteremia

Log-phase cells (BHI; 37° C.) were cultured from master cell banks,sonicated and quantified by spectrophotometry to a desired CFU.

Antimicrobial Agents:

RP-1 (pI/Mw: 10.82/2162.78 Da) is an 18-amino synthetic peptide derivedfrom PF-4 family kinocidins. γ-RP-1 (pI/Mw: 10.64/4444.5 Da) is a39-amino synthetic peptide comprising a CXCL4 kinocidin γ-coremotif-RP-1 construct. Stock peptides dissolved in sterile ddH₂O werediluted in suitable buffers. Antibiotics were purchased as purifiedpowder and re-suspended in PBS per the manufacturer guidelines.

Radial Assay:

RP-1 and γ-RP-1 efficacy was evaluated by radial diffusion at pH 5.5 or7.5. Log-phased organisms were inoculated in buffered agarose andplated. Study peptides (10 μg/well) were introduced into wells in seededmatrix and incubated (3 hr, 37° C.). After overlay of nutrient medium(TSA), the assays were incubated (24 hr) and zones of inhibition definedas radius (mm) of complete clearance minus the well radius. Independentminimum, n=2.

CLSI Assay:

RP-1 and γ-RP-1 were evaluated for their minimum inhibitoryconcentrations (MIC) per CLSI standards in MHB across a 100-0.19 μg/mlrange. Independent minimum, n=2.

Biomatrix Assay:

Efficacies of RP-1 and γ-RP-1 in human blood, plasma, or serum wereassessed. Peptide was added to biomatrix simultaneously with MDRAB(final inoculum 10⁵ CFU/ml; peptide range 1-50 μg/ml; volume 100 μl).Controls were performed using Mueller Hinton broth (MHB; CLSI).Following incubation (constant agitation; 2 h, 37° C.), survival wasquantified by culture (n=3) upon blood agar and defined as CFU/ml.Independent minimum, n=2.

Mechanisms of Action.

Peptide or antibiotic mechanisms versus MDRAB were studied by flowcytometry: 1) forward scatter (FSC; osmostasis); 2) side scatter (SSC;cytoplasmic condensation); 3) energetics (ENR; cytoplasmic membrane [CM]potential); 4) CM permeabilization (PRM); 5) phosphatidylserineaccessibility (PSA; lipid turn over associated with autolysis); and 6)caspase- and/or metacaspase-like activation (CSP; programmed celldeath).

Neutropenic Pneumonia.

Neutropenic mice (cortisone acetate [250 mg/kg; subQ]+cyclophosphamide[200 mg/kg; IP] on study days-2, +3, and +8) were infected on day 0(aerosolized; MDRAB clinical isolate HUMC-1; 5×10⁸ CFU; lungCx-positive). Treatment initiated 4 hr or 24 hr post-infection andcontinued for 3 d. Groups:control (PBS); imipenem (15 mg/kg; IP; bid);γ-RP-1 (12.5 mg/kg; IV; qd); γ-RP-1 (12.5 mg/kg; IV; qd). Survival wasthen monitored to day 21. At end point or 21 d, lungs and spleens werecultured. Outcomes were compared by non-parametric analysis. All studiesadhered to institutional and AAALAC animal care policies.

Results

Control or imipenem-treated mice had 0% or 16% survival, respectively(FIG. 6). RP-1 or γ-RP-1 treatment alone had 28% (p<0.01 vs. control) or72% (p<0.01 vs. control or imipenem) survival, respectively (FIG. 6).The combinations of RP-1 or γ-RP-1 with imipenem treatment achievedrespective 60% or 75% survival rates (p<0.01 vs. control or imipenem,FIG>6). Total MDRAB burden (CFU) in lungs and spleens were significantlyreduced (>2 log CFU) in γ-RP-1 and combination treatment groups ascompared to control or imipenem treatments alone.

FIGS. 2-4 and 6-7 show that RP-1 and γ-RP-1 have significant efficacyvs. MDRAB in human blood ex vivo and in a murine model of neutropenicpneumonia. RP-1 and γ-RP-1 mechanisms of action versus MDRAB involvemultiple targets, including cell membrane perturbation, energydysfunction and programmed cell death (FIG. 5).

The systemic (IV) administration of γ-RP-1 alone or either kinocidin incombination with imipenem achieved robust efficacy vs. MDRAB pneumoniain an otherwise lethal neutropenic murine model. These outcomes affirmRP-1 and γ-RP-1 as innovative and efficacious biologic candidates forfurther evaluation to address the looming threat of MDRAB infections.

The present data further substantiate the γ-RP-1 peptide as overcomingmany historic barriers to anti-infective peptide development, includingsystemic (IV) durability, safety and efficacy in a highly rigorous modelof established infection in a significantly immunocompromised host.

Example 3. RP-1 and γ-RP-1 Efficacy in Mouse Model of MDRPA SkinInfection

In addition to the studies in Example 2 examining systemic efficacy,this example advanced investigations in which RP-1 and γ-RP-1 wereevaluated for topical efficacy in MDRPA skin/skin structure infection(SSSI).

This example first assessed several topical vehicles, and identified 20%P188 poloxamer as appropriate for pre-clinical research, having beentested in humans, low cost, excellent solubility of peptides, rapidabsorption, and no inhibition of peptide antimicrobial activity. In thismurine SSSI model, flanks were shaved and MDRPA (6×10⁵ CFU; log phase)introduced subcutaneously 4 or 24 hr prior to initiation of peptidetreatment. To address any potential immunostimulation effects that maybe caused by the peptides, a strength of the model treats one flank withvehicle alone, and the contralateral flank of the same animal withvehicle+peptide (100 μg). Preliminary data demonstrated strong efficacyin reduction of lesion severity and CFU/abscess, with no apparent skintoxicity due to either peptide.

Pilot studies were also conducted to assess peptide efficacy in SSSI dueto methicillin-resistant S. aureus (MRSA). First, the vehicle (20% P188poloxamer) did not inhibit metapeptide activity vs. a prototype MRSAstrain that caused significant human SSSI, namely LAC-USA300. Comparedto PBS, the vehicle allowed excellent solubility and did not inhibitanti-MRSA activity of either RP-1 or g-RP-1. Next, this example testedefficacy of topical formulations of the peptides in pilot studies usingthe same model system as above (MRSA inoculum, 3×10⁷ CFU). Resultsdemonstrated that the peptides exerted significant efficacy insuppressing MRSA lesion severity over 7 days.

Example 4. Efficacy of RP-1 and γ-RP-1 in a Wide Panel of Gram-NegativePathogens

The efficacy of RP-1 and γ-RP-1, along with a number of peptide variantsand suitable control, was tested against a wide panel of Gram-negativehuman pathogens. The peptides are listed in the table below.

Peptide Remark RP-1 RP-1 (SEQ lD NO: 3) γ-RP-1 γ-RP-1 (SEQ ID NO: 1)IL-8-α α-helix of human IL-8 (KENWVQRVVEKFLKRAENS, SEQ ID NO: 9) γ-IL-8αγ IL-8α (ANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS, SEQ ID NO: 10) PMP-2Comparative rabbit CXCL4 template domain(NLIATKKNGRKLCLDLQAALYKKKIIKKLLES, SEQ ID NO: 11) hIL-8-γγ core of human IL-8 (ANTEIIVKLSDGRELCLDP, SEQ ID NO: 12) ddH₂O control

The following table lists the organisms tested:

Peptide Remark USA300 LAC-USA300 MRSA control E.a. Enterobacteraerogenes K.p. Klebsiella pneumoniae P.m. Proteus mirabilis S.m.Serratia marcenscens E.c. Escherichia coli S.t.R Sal. typhimurium14028-R St.S Sal. typhimurium MS5996-S

The testing results (at pH 5.5 and 7.5) are shown in FIG. 8-11. Theresults demonstrate that γ-RP-1 has the overall best efficacy againstindividual organisms, and across the broader panel, as compared to anyother peptide tested in this study series.

Example 5. In Vitro and In Vivo Anti-Malaria Activity of γ-RP-1

This example tested the anti-malaria activity of γ-RP-1.

FIG. 12 shows the in vitro antimalarial effects of γ-RP-1 on 3D7 P.falciparum. The dose-response curve indicates excellent IC₅₀ curve invitro at 12.5 mg/ml after incubation for 96 hour (two experiments intriplicate). FIG. 13 shows the results of an in vitro toxicity assay ofγ-RP-1 on Human Brain Endothelial Cells (HBECs). This chart shows lowtoxicity for HBECs in vitro at 12.5 mg/ml after incubation for 96 hours(two experiments in triplicate).

In in vivo experiments, FIG. 14 presents images and bar graphs to showthe moderate antimalarial activity in vivo at 12.5 mg/kg for γ-RP-1compared to chloroquine at the same concentration (five mice in eachgroup, IV treatment daily for 5 days). FIGS. 15 and 16 show the in vivoantimalarial activity of γ-RP-1 on Luciferase-expressing P. berghei ANKA(FIG. 15: single dose; FIG. 16: double dose). CQ: chloroquine.

These data demonstrate γ-RP-1's efficacy in treating chloroquine- orartemisinin-resistant malaria.

Example 6. Efficacy of γ-RP-1 and its Variants in a Wide Panel ofGram-Negative Pathogens

The efficacy of RP-1 and γ-RP-1, along with a number of peptide variantsand suitable control, was tested against a wide panel of humanpathogens. The peptides are listed in the table below.

Peptide Remark RP-1 RP-1 (SEQ ID NO: 3) g-RP-1 γ-RP-1 (SEQ ID NO: 1)g-A-RP-1 CPTAQLIATLKNGRKICLDLQAALYKKFKKKLLKSLKRLG (SEQ ID NO: 13)g-P-RP-1 CPTAQLIATLKNGRKICLDLQPALYKKFKKKLLKSLKRLG (SEQ ID NO: 14)17P-g-RP- CPTAQLIATLKNGRKIPLDLQALYKKFKKKLLKSLKRLG 1 (SEQ ID NO: 15)ddH₂O control

As compared to γ-RP-1 (g-RP-1), g-A-RP-1 includes an additional alanineat the C-terminus of the γ-core, g-P-RP-1 includes an additional prolineat the C-terminus of the γ-core, and 17P-g-RP-1 replaces Cys17 of theγ-core with a proline.

The testing results (at pH 5.5 and 7.5) are shown in FIG. 16A-B. Theresults demonstrate that γ-RP-1, as long as its variants g-A-RP-1,g-P-RP-1, and 17P-g-RP-1, all exhibited higher efficacy than RP-1,across bacterial species.

In another experiments, the following peptides were tested.

Peptide Remark g-RP-1 γ-RP-1 (SEQ ID NO: 1) g-RP-1 γ-RP-1 (SEQ ID NO: 1)(genscript) g-AP-RP-1 CPTAQLIATLKNGRKICLDLQAPALYKKFKKKLLKSLKRLG (SEQ ID NO: 16) 14P-g-RP-1 CPTAQLIATLKNGPKICLDLQALYKKFKKKLLKSLKRLG(SEQ ID NO: 17) ddH₂O control

The testing results (at pH 5.5 and 7.5) are shown in FIG. 17A-C, whichshow that all of these variants were active against a spectrum ofbacterial species.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

The disclosures illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising”, “including,” “containing”, etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this disclosure. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the disclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

What is claimed is:
 1. An isolated peptide comprising: (1) a firstfragment consisting of the amino acid sequence of SEQ ID NO:2(CPTAQLIATLKNGRKICLDLQ) or a first amino acid sequence having at least85% sequence identity to SEQ ID NO: 2; and (2) a second fragmentconsisting of the amino acid sequence of SEQ ID NO:3(ALYKKFKKKLLKSLKRLG) or a second amino acid sequence having at least 85%sequence identity to SEQ ID NO: 3, wherein the first fragment and thesecond fragment are connected directly or connected through a spacerthat is 10 amino acids or fewer in length, and wherein the firstfragment is at the N-terminal end of the second fragment.
 2. Theisolated peptide of claim 1, wherein the first fragment and the secondfragment are connected directly.
 3. The isolated peptide of claim 1,wherein the first fragment and the second fragment are connected throughthe spacer.
 4. The isolated peptide of claim 3, wherein the spacer is 5amino acids or fewer in length.
 5. The isolated peptide of claim 1,comprising the amino acid sequence of SEQ ID NO:1(CPTAQLIATLKNGRKICLDLQALYKKFKKKLLKSLKRLG).
 6. The isolated peptide ofclaim 1, comprising an amino acid sequence having at least 90% sequenceidentity to SEQ ID NO:1.
 7. The isolated peptide of claim 1, comprisingan amino acid sequence having from one to five amino acid substitutionsfrom SEQ ID NO:1.
 8. The isolated peptide of claim 1, wherein thepeptide is not longer than 100 amino acids in length.
 9. The isolatedpeptide of claim 8, wherein the peptide is not longer than 75 aminoacids in length.
 10. The isolated peptide of claim 8, wherein thepeptide is not longer than 50 amino acids in length.
 11. The isolatedpeptide of claim 1, wherein the peptide has antimicrobial activity. 12.A polynucleotide comprising a nuclei acid sequence encoding the peptideof claim
 1. 16. A composition comprising the peptide of claim 1 and apharmaceutically acceptable carrier.
 17. A method of treating aninfection in a patient in need thereof, comprising administering to thepatient an effective amount of the composition of claim
 16. 18. Themethod of claim 17, wherein the infection is caused bymultidrug-resistant Pseudomonas aeruginosa (MDRPA) ormultidrug-resistant Acinetobacter baumannii (MDRAB).
 19. The method ofclaim 17, wherein the infection is caused by a parasite.
 20. The methodof claim 19, wherein the patient suffers from malaria.